Treatment of Cancer WIth Dopamine Receptor Antagonists

ABSTRACT

Described are methods of treating a cancer comprising administering to a subject in need thereof an effective amount of a dopamine receptor (DR) antagonist. The DR antagonist may be a phenothiazine derivative, such as thioridazine or chlorpromazine. Optionally, the cancer is acute myeloid leukemia. Also described are methods for identifying subjects with cancer, methods for providing a prognosis for a subjects with cancer and/or subjects likely to be responsive to therapy with DR receptor antagonists. Methods for identifying cancer stem cells and chemotherapeutic compounds that are DR receptor antagonists as also provided.

RELATED APPLICATIONS

This application is a 35 USC 371 National Stage Entry ofPCT/CA2012/000175 filed on Feb. 28, 2012, which claims the benefit ofU.S. Provisional Application No. 61/447,362, filed on Feb. 28, 2011 eachof which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates to methods for the prognosis or treatment ofcancer and particularly to methods for the prognosis or treatment ofacute myeloid leukemia (AML) that target dopamine receptors.

BACKGROUND OF THE DISCLOSURE

Increasing evidence suggests that cancer/tumor development is due to arare population of cells, termed cancer stem cells (CSCs) (Dick, 2009;Jordan, 2009; Reya et al., 2001) that are uniquely able to initiate andsustain disease. In addition, experimental evidence indicates thatconventional chemotherapeutics, characterized by their ability toinhibit cell proliferation of cancer cell lines (Shoemaker, 2006) orreduce tumor burden in murine models (Frese and Tuveson, 2007), areineffective against human CSCs (Guan et al., 2003; Li et al., 2008).This resistance to chemotherapeutics is coupled with indiscriminatecytotoxicity that often affects healthy stem and progenitor cells,leading to dose restriction and necessitating supportive treatment(Smith et al., 2006). Recent examples along these lines includeselective induction of apoptosis (Gupta et al., 2009; Raj et al., 2011)that remains to be tested in normal SCs and in the human system.Accordingly, the identification of agents that target CSCs alone is nowcritical to provide truly selective anti-cancer drugs for pre-clinicaltesting.

Normal and neoplastic SCs are functionally defined by a tightlycontrolled equilibrium between self-renewal vs. differentiationpotential. In the case of CSCs, this equilibrium shifts towards enhancedself-renewal and survival leading to limited differentiation capacitythat eventually allows for tumor growth. In contrast to direct toxiceffects that equally affect normal SCs, an alternative approach toeradicate CSCs is by modification of this equilibrium in favor ofdifferentiation in an effort to exhaust the CSC population. Theidentification of molecules that selectively target somatic CSCs whilesparing healthy SC capacity would therefore be useful for thedevelopment of novel diagnostics and therapeutic treatments toselectively target human CSCs.

Hematological malignancies are types of cancer that affect blood, bonemarrow and lymph nodes. Hematological malignancies may derive fromeither of the two major blood cell lineages: myeloid and lymphoid celllines. Examples of myeloid malignancies include acute myeloid leukemiaand chronic myeloid leukemia.

While myeloid malignancies are all generally considered to arise fromprecursors of the myeloid lineage in the bone marrow, they are highlydivergent in presentation, pathology and treatment. For example, the2008 World Health Organization Classification for MyeloproliferativeNeoplasms (See Tefferi et al. Cancer, September 1^(st), pp. 3842-3847(2009); also Vannucchi et al. Advances in Understanding and Managementof Myeloproliferative Neoplasms CA Cancer J. Clin. 2009; 59:171-191,both hereby incorporated by reference), identifies 5 differentclassification schemes for myeloid neoplasms, and places acute myeloidleukemia (AML) in a separate category from chronic myelogenous leukemia(CML) and other myeloproliferative neoplasms. Furthermore, CML is oftencharacterized as containing the BCR-Abl translocation which is absent inAML. Preferred treatments for leukemias, such as myeloid malignancies,would target leukemic cells without unduly affecting hematopoietic stemcell populations.

Thioridazine is a dopamine receptor antagonist that belongs to thephenothiazine drug group and is used as an anti-psychotic. It has beenin clinical use since 1959, however because of concerns aboutcardiotoxicity and retinopathy at high doses this drug is not commonlyprescribed, and is reserved for patients who have failed to respond to,or have contraindications for more widely used antipsychotics.Schizophrenic patients receiving dopamine receptor antagonist medicationat doses deemed effective for schizophrenia have been reported to have areduced incidence of rectum, colon, and prostate cancer compared to thegeneral population.

There is a need for novel methods for the treatment and prognosis ofcancers and in particular for novel methods for the treatment andprognosis of acute myeloid leukemia.

SUMMARY OF THE DISCLOSURE

It has surprisingly been determined that dopamine receptor antagonistssuch as thioridazine or chlorpromazine are cytotoxic to cancer cells andin particular acute myeloid leukemia (AML). Furthermore, dopaminereceptors antagonists at concentrations toxic to cancer cells have beenfound to have a relatively limited effect on normal stem cells such ashematopoietic stem cells. It has also been determined that dopaminereceptors are expressed in AML cell lines and in primary AML cells, butshow relatively less expression in cell lines enriched for normalhematopoietic stem cells. In addition, the expression of dopaminereceptors in AML cells is shown to correlate with that of themonoblastic marker CD14. Dopamine receptor antagonists such asthioridazine are cytotoxic to AML cells that express CD14.

Accordingly, in one aspect there is provided a method of treating acancer or precancerous disorder in a subject comprising administering tothe subject in need thereof a dopamine receptor (DR) antagonist. In asimilar aspect, the present disclosure describes the use of a dopaminereceptor antagonist for the treatment of cancer or a precancerousdisorder. In one embodiment, the cancer or precancerous disorder is amyeloproliferative disease or leukemia. In one embodiment, the cancer isacute myeloid leukemia (AML). In one embodiment, the DR antagonistpreferentially induces the differentiation of cancer stem cells relativeto hematopoietic or normal stem cells.

In one embodiment, the dopamine receptor antagonist is a phenothiazinederivative such as thioridazine or chlorpromazine. Optionally thedopamine receptor antagonist is a multi-receptor antagonist thatantagonizes more than one dopamine receptor. In one embodiment, thedopamine receptor antagonist is a D₂ family dopamine receptorantagonist. In one embodiment, the DR antagonist is a compound selectedfrom those listed in Table 1.

In another aspect there is provided a method for reducing theproliferation of a cancer cell comprising contacting the cancer cellwith a dopamine receptor antagonist. In a similar aspect there isprovided the use of a dopamine receptor antagonist for reducing theproliferation of a cancer cell. In one embodiment, contacting the cellwith a dopamine receptor antagonist induces cell death ordifferentiation of a cancer cell or precancerous cell. In oneembodiment, the cancer cell is a cancer stem cell and contacting thecancer stem cell with a dopamine receptor antagonist inducesdifferentiation of the cancer stem cell. The cell may be in vivo or invitro. In one embodiment, the precancerous cell is a myeloproliferativecell. Optionally, the cancer cell is a leukemic cell, such as an acutemyeloid leukemia (AML) cell or a monocytic leukemic cell. In oneembodiment, the cell is CD14 positive. In one embodiment, the dopaminereceptor antagonist is a phenothiazine derivative such as thioridazine.In one embodiment, the dopamine antagonist is a compound selected fromthose listed in Table 1.

In another aspect there is provided a method of identifying a subjectwith cancer suitable for treatment with a dopamine receptor antagonist.In one embodiment, the method includes determining the expression of oneor more dopamine receptors in a sample of cancer cells from the subject.In one embodiment, subjects with cancer cells that express one or moredopamine receptors are identified as suitable for treatment withdopamine receptor antagonists. In one embodiment, the cancer is leukemiaand the cancer cells are leukemic cells. In one embodiment, the leukemiais acute myeloid leukemia or monocytic leukemia. In one embodiment, thecancer is breast cancer. In one embodiment, the method of identifying asubject with cancer comprises testing the sample for the expression ofCD14.

In one aspect, there is provided a method for determining a prognosisfor a subject with cancer, comprising determining the expression levelof one or more dopamine receptor biomarkers in a sample from the subjectand comparing the level of expression of the one or more biomarkers to acontrol. Optionally, the method provided herein include providing orobtaining a sample of cancer cells from the subject. In one embodiment,increased expression of one or more biomarkers compared to the controlindicates a more severe form of cancer. In one embodiment, the dopaminereceptor biomarkers are DR3 and/or DR5. In one embodiment, the cancer isleukemia or breast cancer and the sample comprises leukemic cells orbreast cancer cells. In one embodiment, the leukemia is acute myeloidleukemia or monocytic leukemia.

Also provided are methods for identifying a subject with leukemia. Inone embodiment, the methods include determining the expression level ofone or more dopamine receptors in a sample from the subject andcomparing the level of expression of the one or more dopamine receptorsto a control. Optionally, sample comprises white blood cells and/or themethod further comprises providing a sample comprising white blood cellsfrom the subject. In one embodiment, increased expression levels of oneor more dopamine receptors compared to a control is indicative of asubject with leukemia, such as acute myeloid leukemia, or monocyticleukemia. In one embodiment, the method further comprises testing forCD14.

Also provided are methods for screening compounds for anti-canceractivity comprising identifying compounds that are dopamine receptorantagonists. In one embodiment, the anti-cancer activity is reducedproliferation of AML cells or monocytic cells. Optionally, the methodsinclude identifying compounds that preferentially induce thedifferentiation of cancer stem cells relative to hematopoietic or normalstem cells.

In one aspect, there is provided methods for identifying a cancer stemcell from a population of cells. In one embodiment, the method comprisesdetermining whether a cell expresses one or more biomarkers selectedfrom dopamine receptor (DR) 1, DR2, DR3, DR4 and DR5. In one embodiment,the expression of dopamine receptor (DR) 1, DR2, DR3, DR4 and DR5 isindicative that the cell is a cancer stem cell.

In one embodiment, the population of cells comprises cells isolated froma mammal or cells in culture such as cell culture. In one embodiment,the population of cells comprises pluripotent stem cells. In oneembodiment, the population of cells comprises cancer cells such ashematological cancer cells or pre-cancerous cells. Optionally, themethod includes testing the cell for the expression of polynucleotidesor polypeptides that code for DR1, DR2, DR3, DR4 or DR5. In oneembodiment, a cell that expresses DR1, DR2, DR3, DR4 and DR5 isidentified as a cancer stem cell. In some embodiments, the methodsdescribed herein also include isolating cancer stem cells from apopulation of cells. For example, cells that are identified as cancerstem cells can be isolated from a population of cells or other materialusing methods known in the art such as flow cytometry, fluorescenceactivated cell sorting, panning, affinity column separation, or magneticselection. In one embodiment, cancer stem cells are isolated usingantibodies to one or more of DR1, DR2, DR3, DR4 and DR5.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments of the disclosure will now be described inrelation to the drawings in which:

FIG. 1 shows thioridazine at 10 μM is cytotoxic to leukemic cell linesHL-60, MV4-11 and OCI3.

FIG. 2 shows that thioridazine 10 μM has limited affects on the colonyforming potential of normal HSCs (2A) while significantly reducing AMLblast forming potential.

FIG. 3 shows cell pellets of CFU colonies generated from normal HSC andAML treated with Thioridazine.

FIG. 4 shows that both 10 μM chlorpromazine and 10 μM thioridazine iscytotoxic to leukemic cell lines HL-60, MV4-11 and OCI3.

FIG. 5 shows the expression of dopamine receptors DR1, DR2, DR3, DR4 andDR5. DR expression was observed in AML cell lines, some primary AML andmononuclear cells (MNC) but not in HSC enriched cells (CB lin(−)).

FIG. 6 shows that multiple DR antagonists are cytotoxic to AML celllines. SKF=(R)-(+)-SKF-38393 hydrochloride; 7OH=R(+)-7-Hydroxy-DPAThydrobromide; GR=GR 103691; SCH=R(+)-SCH-23390 hydrochloride;CLOZ=Clozapine; CHL=Chlorpromazine hydrochloride; THIO=Thioridazine.

FIG. 7 shows FACS data showing that dopamine receptors are expressed inthe population of CD14+ cells in primary AML.

FIG. 8 shows that thioridazine selectively targets and reduces thenormalized frequency of CD14+ cells in primary AML.

FIG. 9 shows the identification of mefloquine and thioridazine usingchemical screening for compounds that differentiate neoplastic hPSC. (A)Schematic of screening strategy. (B) XY-scatter plot of percent residualactivity (% RA) of GFP and Hoechst signals of the 590 compound screen.Region outlined demonstrates loss of pluripotency (LOP) as defined byreduced GFP and Hoechst. Each point n=3, mean+/−SD (C) Summary ofresponses seen with 590 compounds. (D) Chemical structure of candidatecompounds; thioridazine, azathioprine and mefloquine. (E) RepresentativeGFP, Hoechst and merged microscopic images of v1H9-Oct4-GFP cellstreated with candidate compounds at 10 μM. (F) Histogram of GFPintensity of these images. (G) Dose response curves of v1H9-Oct4-GFPtreated with candidate compounds and calculation of EC₅₀. Each pointn=3; mean+/−SEM.

FIG. 10 shows the effect of salinomycin, mefloquine and thioridazine onnormal and neoplastic populations. (A-B) Flow cytometry analysis offrequency of Oct4+ cells in (A) H9 and (B) v1H9-Oct4-GFP cells treatedwith salinomycin (SAL), mefloquine (MQ) and thioridazine (THIO) at10⁻⁷-10⁻⁶M. Each bar n=3; mean+/−SD. Values are normalized toDMSO-treated control samples; (—) DMSO mean, (- -) mean minus one SD,(—) level of % Oct4+ in BMP4 treated samples. (C) Ratio of normalized %Oct4+ cells in H9 per v1H9-Oct-GFP with same compound at the sameconcentration. Percent of neoplastic hPSC staining positive for (D) p53and (E) p21 following 24 h treatment with 10 μM etoposide, 10 μMthioridazine (THIO), BMP4 and DMSO-treated (CTRL) controls. Each barn=3; mean+/−SD. Representative images of etoposide and thioridazinetreated cells included. Arrows show p53+ and p21+ in etoposide-treatedcells versus thioridazine-treated cells. (F) Differentiation-associatedgenes with >2 fold increase following thioridazine treatment ofneoplastic hPSC. Genes divided into respective lineages, endoderm(ENDO), mesoderm (MESO), germ cell (GERM), neural (NEURO) andtrophoblast (TROPH). Each bar represents the mean of two separateexperiments. (G-K) Hematopoietic multilineage and clonogenic potentialin response to compound treatment detected using methycellulose assays.Representative colony forming unit (CFU) pellets of (G) hematopoieticstem and progenitor cells (HSPC) versus (H) AML blast CFUs pelletsfollowing compound treatment. (I-J) Quantification of respective CFUsand blast-CFUs generated from (I) HSPC and (J) AML blast cells followingcompound treatment. Values were normalized to DMSO-treated controlsamples; (—) DMSO mean, (- -) mean minus one SEM. Each HSPC bar n=7individual samples, mean+/−SEM. Each AML bar at least n=5 individualpatient samples, mean+/−SEM. (K) Ratio of normalized HSPC CFUs per AMLblast CFUs with same compound at the same concentration. (L) Frequencyof normalized CD11b granulocytic cells in cultured patient AML cellstreated with thioridazine 10 μM (THIO 10 μM) or DMSO vehicle (CTRL) forup to 96 hours. Each bar n=3, mean+/−SD. (*) p<0.05, (**) p<0.01, (***)p<0.001, (****) p<0.0001.

FIG. 11 shows the effect of salinomycin, mefloquine and thioridazine onfibroblast-derived iPSC and HSPC. (A) Flow cytometry analysis offrequency of Oct4+ cells in fibroblast-derived iPSC (Fib-iPS) treatedwith salinomycin (SAL), mefloquine (MQ) and thioridazine (THIO) at10⁻⁷-10⁻⁶M. Each bar n=3; mean+/−SD. Values are normalized toDMSO-treated control samples; (—) DMSO mean, (- -) mean minus one SD,(—) level of % Oct4+ in BMP4 treated samples. (B) Extended dose responseof compounds on neoplastic hPSC. Each point mean+/−SEM, (C)Hematopoietic lineage potential of CBlin− treated with thioridazine.Colony forming units (CFUs) of erythoblast (CFU-E), macrophage (CFU-M)and granulocyte (CFU-G) colonies generated in methylcellulose assays.(D) Composition of CFU generated from CBlin− treated with salinomycin,mefloquine and thioridazine. Percent composition of CFUs generated withsalinomycin (SAL), mefloquine (MQ) and thioridazine (THIO) treatment at0.1 μM, 1 μM and 10 μM. (*) p<0.05, (**) p<0.01

FIG. 12 shows thioridazine's effect on HSC and LSC engraftment. (A)Frequency of human CD45+ cells in the bone marrow following HSPCtreatment with thioridazine 10 μM (THIO 10 μM) or mefloquine 10 μM (MQ10 μM). Values normalized to DMSO-treated HSPC control (CTRL) samples.Total of two HSPC samples evaluated. Mean+/−SEM. (B) Representative flowcytometry plots of side scatter (SSC) versus myeloid (CD33) or lymphoid(CD19) markers within the hCD45+ population. 12(C) Frequency of CD45+CD33+ AML blast cells in the bone marrow (BM) following treatment of AMLwith thioridazine 10 μM (THIO 10 μM) or mefloquine 10 μM (MQ 10 μM).Values normalized to DMSO-treated AML control (CTRL) samples. Total oftwo AML patient samples evaluated. (D) Representative flow plots of CD33vs CD45 in DMSO-treated control (CTRL) populations versus thioridazinetreated (THIO 10 μM). (E) Ratio of normalized percent hCD45 HSPCengraftment per normalized percent CD45 CD33 AML blast engraftment. (*)p<0.05

FIG. 13 shows in vivo response to drug treatment. (A) The normalizedfrequency of human CD45+ cells in the bone marrow following HSPCtreatment with salinomycin 1 μM (SAL 1 μM) relative to DMSO-treated(CTRL) samples. Total of two HSPC samples evaluated. Mean+/−SEM. (****)p<0.0001 (B) Thioridazine's effect on HSC and LSC splenic engraftment.(B, top) Frequency of human CD45+ cells in the spleen following HSPCtreatment with thioridazine 10 μM (THIO 10 μM). Values normalized toDMSO-treated HSPC control (CTRL) samples. Total of two HSPC samplesevaluated. Mean+/−SEM. (B, bottom) CD45+ CD33+ blast cells in the spleenfollowing thioridazine 10 μM (THIO 10 μM) treatment of AML. Valuesnormalized to DMSO-treated AML control (CTRL) samples. Total of two AMLpatient samples evaluated. (C) Thioridazine's effect on erythrocytic andmegakaryocytic regeneration. Composition of human blood cells detectedin the xenotransplant BM injected with HSPC treated with thioridazine 10μM (THIO 10 μM) or with DMSO (CTRL). Red blood cells (RBC) are definedby glycophorin A positivity and platelets by CD41a. (D) Confirmation ofmyeloid leukemic engraftment of xenotransplants with AML. Flow cytometryof side scatter versus CD19, a marker of lymphoid cells. Inset numberrepresents mean+/−SEM. (E-F) Thioridazine's effect on HSC and LSC invivo self-renewal. Engraftment levels of (E) hCD45+ cells or (F)hCD45+CD33+ in BM of secondary xenotransplants receiving equal number ofhCD45 cells explanted from (E) primary CBlin− or (F) primary AMLtransplants treated with thioridazine (THIO 10 μM) or DMSO control(CTRL). Each bar n=3 mice, mean+/−SEM.

FIG. 14 shows dopamine receptors expressed on neoplastic stem cells.(A-B) Flow cytometry of (A) normal H9 and (B) neoplastic v1H9-Oct4-GFPcells stained with SSEA3 and all five dopamine receptor (DR) subtypes.DR expression in the SSEA3+ fraction is shown. (C) Flow cytometry oflineage-depleted cord blood (HSPC) stained with CD34, CD38 and all fiveDR subtypes. DR expression is presented in the gated populations. (D)Flow cytometry of 13 AML patient samples stained for all five DRs alongwith associated FAB classification. (E) Co-localization of DRD5 intriple-negative (ER−, PR− and HER2−) primary human breast tumor stainedwith CD44 and CD24. (F) The frequency of triple-negative breast CSC(CD44+CD24−/^(lo)) within the DRD3 and DRD5 population. Each barcomposed of 3 primary triple-negative breast tumors, mean+/−SEM. (G-H)Frequency of AML blast cells (CD33+CD45+) from patient samples which arealso positive for (G) DRD3 and (H) DRD5. A total of 8 AML patientsamples were assessed for leukemic-initiation potential inxenotransplantation recipients. Leukemic-initiating was defined as humanengraftment >0.1% of CD33+ hCD45+ in mouse bone marrow. Fourleukemic-initiating AML samples were assayed in 22 mice while 4non-initiating AML samples were assayed in 17 mice. Total n=8 AMLsamples, mean+/−SEM.

FIG. 15 (A-B) Flow cytometry SSEA3+ fraction in (A) fibroblast-derivedhiPSC and (B) umbilical cord blood-derived hiPSC stained for all fivedopamine receptors. (C) Dopamine receptors expression of human bloodpopulations. Flow cytometry of cord blood mononuclear cells stained for(C) erythroid (glycophorin A), (C) megakaryocytes (CD41a); (D) T-cells(CD3), (D) B-cells (CD19); (E) monocytes (CD14) and (E) granulocytes(CD15). Staining for all five DRs in the gated populations are shown ashistograms. (F) Summary of DR localization in the blood populations. (G)Flow cytometry of AML patient showing DR in gated populations. (H)Dopamine receptor expression in triple-negative human breast tumors.Breast CSC are defined as CD44+ CD24−/^(lo) (Al-Hajj et al., 2003).Co-localization of each DR within the CD44 and CD24 population is shownfor three triple-negative (ER−, PR− and HER2−) breast tumors.

FIG. 16 shows that thioridazine inhibits dopamine receptor signalling inAML. (A) DR expression of AML-OCI2 and AML-OCI3 cell lines. (B) Cellcounts of AML-OCI2 and AML-OCI3 cells treated with three DR antagonistdrugs. Values are normalized to DMSO-treated control samples. Each barn=3; mean+/−SD. (C-D) Viable cell counts (7AAD−, Hoechst+) of same celllines treated with (C) 70H-DPAT, a DR D2-family agonist, or (D)SKF38393, a DR D1-family agonist, in serum-free conditions. Values arenormalized to DMSO-treated control samples. Each bar n=3; mean+/−SD.

DETAILED DESCRIPTION I. Definitions

As used herein, the term “cancer” refers to one of a group of diseasescaused by the uncontrolled, abnormal growth of cells that can spread toadjoining tissues or other parts of the body. Cancer cells can form asolid tumor, in which the cancer cells are massed together, or exist asdispersed cells, as in leukemia.

The term “cancer cell” as used herein refers a cell characterized byuncontrolled, abnormal growth and the ability to invade another tissueor a cell derived from such a cell. Cancer cell includes, for example, aprimary cancer cell obtained from a patient with cancer or cell linederived from such a cell. Similarly, a “hematological cancer cell”refers to a cancer cell deriving from a blood cell or bone marrow cell.Examples of cancer cells include, but are not limited to, cancer stemcells, breast cancer cells, rectum cancer cells, colon cancer cells,prostate cancer cells and hematological cancer cells such as myelomas,leukemic cells or lymphoma cells.

As used herein the term “cancer stem cell” refers to a cell that iscapable of self-renewal and differentiating into the lineages of cancercells that comprise a tumor or hematological malignancy. Cancer stemcells are uniquely able to initiate and sustain the disease.

The term “precancerous disorder” as used herein refers to one of a groupof hyperproliferative disorders that can develop into cancer, includingfor example precancerous blood disorders, such as myeloproliferativedisease or myelodysplastic syndrome which is a premalignant conditionthat is related to and/or can develop into acute myeloid leukemia (AML).

The term “precancerous cell” as used herein refers to a cellcharacterized by uncontrolled, abnormal growth or a cell derived fromsuch a cell. The term “precancerous cell” includes, for example, aprimary precancerous cell obtained from a patient with precancerousdisorder or cell line derived from such a cell or a cancer stem cell.Similarly, a “hematological precancerous cell” refers to a precancerouscell deriving from a blood cell or bone marrow cell. In one embodiment,the hematological precancerous cell is a myeloproliferative cell.

The term “leukemia” as used herein refers to any disease involving theprogressive proliferation of abnormal leukocytes found in hemopoietictissues, other organs and usually in the blood in increased numbers.“Leukemic cells” refers to leukocytes characterized by an increasedabnormal proliferation of cells. Leukemic cells may be obtained from asubject diagnosed with leukemia.

The term “acute myeloid leukemia” or “acute myelogenous leukemia”(“AML”) refers to a cancer of the myeloid line of blood cells,characterized by the rapid growth of abnormal white blood cells thataccumulate in the bone marrow and interfere with the production ofnormal blood cells. Pre-leukemic conditions such as myelodysplastic ormyeloproliferative syndromes may also develop into AML.

As used herein, the term “monocytic leukemia” refers to a subtype ofleukemia characterized by the expression of CD14, and includes AcuteMonocytic Leukemia, which is a subtype of acute myeloid leukemia. In oneembodiment, a subject is identified as having acute monocytic leukemiaif they have greater than 20% blasts in the bone marrow, and of these,greater than 80% are of the monocytic lineage.

The term “dopamine receptor antagonist” refers to a compound thatproduces any detectable or measurable reduction in the function oractivity of one or more dopamine receptors. In one embodiment, thedopamine receptors (DR) are selected from DR1, DR2, DR3, DR4 and DR5.Dopamine receptor antagonists may be selective for one or multipledopamine receptors, i.e. a “multi-receptor antagonist”. Examples ofmulti-receptor dopamine antagonists include thioridazine andchlorpromazine. Dopamine receptors are commonly grouped in D₁-familydopamine receptors (DR1 and DR5) and D₂-family dopamine receptors (DR2,DR3 and DR4). In one embodiment, the dopamine receptor antagonist is acompound selected from those listed in Table 1.

TABLE 1 Dopamine antagonists suitable for use in the methods describedherein. Dopamine Receptor Antagonist Mechanism of Action Acetopromazinemaleate salt Dopaminergic antagonist Amisulpride D2 and D3 receptorantagonist Amoxapine Dopamine-reuptake inhibitor Azaperone Dopaminergicreceptor antagonist Benperidol Dopamine antagonistBenzo[a]phenanthridine-10,11-diol, D1 ligand Bromopride Dopamineantagonist Bromperidol Dopamine antagonist Chlorpromazine hydrochlorideD2 antagonist, selective D1, D3, D4 & D5 Chlorprothixene hydrochlorideD2 dopamine receptor antagonist Clomipramine hydrochloridechlorpromazine derivative Disulfiram Dopamine beta-hydroxylase inhibitorDO 897/99 D3 antagonist Domperidone Dopamine Antagonists DROPERIDOL D2(dopamine receptor) antagonist Ethopropazine hydrochloride Thioridazinederivative Fluperlapine D2 (dopamine receptor) antagonist Fluphenazinedihydrochloride Dopamine antagonist D1 &D2 antagonist GBR 12909dihydrochloride Dopamine reuptake inhibitor Haloperidol Dopamineantagonist D2, non-selective antagonist Hydrastinine hydrochlorideDopamine receptor blocker Indatraline potent D antagonist ItoprideDopamine D2 receptors and ACE inhibition LEVOSULPIRIDE D2, D3, & D4antagonist Loxapine succinate Dopamine antagonist/D2, D4 Mesoridazine D2antagonist Mesoridazine besylate D antagonist Methotrimeprazine maleatsalt Thioridazine derivative Metixene hydrochloride Thioridazinederivative Molindone hydrochloride Dopamine receptor antagonistNafadotride D3 antagonist Nomifensine maleate Dopamine uptake inhibitorOLANZAPINE D1&D2 antagonist PEROSPIRONE HCl D2&D4 antagonistPerphenazine D1 & D2 antagonist PHENOTHIAZINE Thioridazine derivativePimozide Dopamine antagonist Piperacetazine Thioridazine derivativeProchlorperazine Thioridazine derivative Prochlorperazine dimaleateDopamine antagonist Promazine hydrochloride Dopamine receptor antagonistPromethazine hydrochloride Thioridazine derivative Quetiapine dopamineand serotonin receptors antagonist QUETIAPINE HEMIFUMARATE D2 antagonistR(+)-SCH-23390 hydrochloride D1 antagonist Raclopride D2 antagonistRemoxipride Hydrochloride Dopaminergic antagonist RISPERIDONE D1 & D2antagonist S(−)Eticlopride hydrochloride Dopamine receptor antagonistSertindole Dopamine D2/Serotonin 5-HT2 receptor antagonist SKF 83566 D1antagonist Spiperone D2 antagonist Sulpiride D2 antagonist Sulpiride D2& D3 antagonist Thiethylperazine malate Thioridazine derivativeThioproperazine dimesylate D1 & D2 antagonist Thioridazine hydrochlorideThioridazine derivative TRIFLUOPERAZINE D2 antagonist Triflupromazinehydrochloride D1 & D2 antagonist Trimeprazine tartrate Thioridazinederivative Trimethobenzamide hydrochloride D2 antagonist ZiprasidoneHydrochloride Dopamine D2/serotonin 5-HT2 antagonist Zotepine DopamineD2/serotonin 5-HT2 antagonist

As used herein, the term “phenothiazine” or “phenothiazine derivative”refers to a compound that is derived from or contains a phenothiazinemoiety or backbone. Phenothiazine has the formula S(C₆H₄)₂NH andphenothiazine derivatives comprise one or more substitutions oradditions to phenothiazine. For example, some phenothiazine derivativeshave a three-ring structure in which two benzene rings are linked by anitrogen and a sulfur. Examples of phenothiazine derivatives includethioridazine, chlorpromazine, levomepromazine, mesoridazine,fluphenazine, perphenazine, prochlorperazine, and trifluoperazine.Additional examples of phenothiazine derivatives for use in the methodsof the present disclosure are set out in Table 1. In one embodiment,thioridazine has the IUPAC name10-{2-[(RS)-1-Methylpiperidin-2-yl]ethyl}-2-methylsulfanylphenothiazine.Optionally, one or more racemic forms of a phenothiazine derivative suchas thioridazine are used in the methods described herein.

As used herein, “reducing the proliferation of a cancer cell” refers toa reduction in the number of cells that arise from a cancer cell as aresult of cell growth or cell division and includes cell death ordifferentiation of a cancer stem cell. The term “cell death” as usedherein includes all forms of cell death including necrosis andapoptosis. As used herein “differentiation of a cancer stem cell” refersto the process by which a cancer stem cell loses the capacity toself-renew and cause the lineages of cancer cells that comprise a tumoror hematological malignancy.

The term “determining a prognosis” refers to a prediction of the likelyprogress and/or outcome of an illness, which optionally includes definedoutcomes (such as recovery, symptoms, characteristics, duration,recurrence, complications, deaths, and/or survival rates).

As used herein the term “control” refers to a comparative sample or apre-determined value. In one embodiment, “control” refers to a level ofexpression of a biomarker as described herein. In one embodiment, thecontrol is representative of normal, disease-free cell, tissue, orblood. In one embodiment, the control is representative of subjects withcancer for whom the clinical outcome or severity of the disease isknown. For example, in one embodiment the “control” is representative ofsubjects who have survived for at least 5 years after a diagnosis withAML. In one embodiment, the “control” is representative of subjects withcancer who have a particular stage of grade of the disease. In oneembodiment, the “control” is representative of stem cells that are notcancer stem cells.

As used herein, the phrase “effective amount” or “therapeuticallyeffective amount” means an amount effective, at dosages and for periodsof time necessary to achieve the desired result. For example in thecontext or treating a cancer such as AML, an effective amount is anamount that for example induces remission, reduces tumor burden, and/orprevents tumor spread or growth of leukemic cells compared to theresponse obtained without administration of the compound. Effectiveamounts may vary according to factors such as the disease state, age,sex and weight of the animal. The amount of a given compound that willcorrespond to such an amount will vary depending upon various factors,such as the given drug or compound, the pharmaceutical formulation, theroute of administration, the type of disease or disorder, the identityof the subject or host being treated, and the like, but can neverthelessbe routinely determined by one skilled in the art.

The term “pharmaceutically acceptable” means compatible with thetreatment of animals, in particular, humans.

The term “subject” as used herein includes all members of the animalkingdom including mammals, and suitably refers to humans. Optionally,the term “subject” includes mammals that have been diagnosed with canceror are in remission.

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e. not worsening) state of disease(e.g. maintaining a patient in remission), preventing spread of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, diminishment of the reoccurrence of disease, andremission (whether partial or total), whether detectable orundetectable. “Treating” and “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.“Treating” and “treatment” as used herein also include prophylactictreatment. In one embodiment, treatment methods comprise administeringto a subject a therapeutically effective amount of a dopamine receptorantagonist as described herein and optionally consists of a singleadministration, or alternatively comprises a series of administrations.

II. Methods and Uses

It has surprisingly been found that dopamine receptor (DR) antagonistsare cytotoxic to AML lines and primary AMLs while being much less toxicto normal hematopoietic stem cells. As shown in Examples 2 and 10, theDR antagonist thioridazine significantly reduced leukemic stem cell(LSC) function while preserving normal hematopoietic stem cell capacity.

Accordingly, in one embodiment there is provided a method of treating acancer or precancerous disorder in a subject comprising administering tothe subject in need thereof a dopamine receptor antagonist. Alsoprovided is a use of a dopamine receptor antagonist for the treatment ofcancer or a precancerous disorder. In one embodiment, the methods oruses described herein are useful to treat a precancerous disorder, suchas a myeloproliferative disease. In one embodiment, the cancer isleukemia such as acute myeloid leukemia (AML), or monocytic leukemia.The methods and uses described herein are particularly useful for thetreatment of cancer cells that express dopamine receptors. In oneembodiment, the methods and uses described herein are useful for thetreatment of cancer cells that express the monocytic marker CD14. In oneembodiment, the dopamine receptor antagonist preferentially induces thedifferentiation of cancer stem cells in the subject relative tohematopoietic or normal stem cells. In one embodiment, the cancer stemcells are leukemic cancer stem cells. In one embodiment, the subject hasAML and the cancer stem cells are AML cancer stem cells.

In one embodiment, the dopamine receptor antagonists are antagonists forone or more of dopamine receptors (DR) such as DR1, DR2, DR3, DR4, andDR5. Optionally the DR antagonist is a multi-receptor antagonist, or isspecific for a single dopamine receptor subtype. In one embodiment, theDR antagonist is a phenothiazine derivative such as thioridazine, orchlorpromazine. In one embodiment, the DR antagonist is selected fromthe compounds listed in Table 1. A person of skill in the art wouldreadily be able to identify additional dopamine receptor antagoniststhat are useful for the treatment of cancer as described herein.

In one embodiment, the methods or uses described herein involve aphenothiazine derivative such as thioridazine or chlorpromazine. Aperson skilled in the art would readily be able to identify additionalphenothiazine derivatives that are dopamine receptor antagonists anduseful for the treatment of cancer as described herein. In oneembodiment, the phenothiazine derivatives have a differential toxicityfor cancer cells, such as leukemic cells, compared to normal stem cellsor hematopoietic stem cells.

In one embodiment, the dopamine receptor antagonists and/orphenothiazine derivatives are prepared for administration to a subjectin need thereof as known in the art. Conventional procedures andingredients for the selection and preparation of suitable formulationsare described, for example, in Remington's Pharmaceutical Sciences(2003—20th edition) and in The United States Pharmacopeia: The NationalFormulary (USP 24 NF19) published in 1999.

In one embodiment, there is also provided a method for reducing theproliferation of a cancer cell or cells comprising contacting the cellwith a dopamine receptor antagonist. In a similar embodiment there isprovided a use of a dopamine receptor antagonist for reducing theproliferation of a cancer cell or cells. In one embodiment, the cancercell is a cancer stem cell. In one embodiment, the DR antagonist inducesdifferentiation or cell death of a cancer stem cell. In one embodiment,the DR antagonist induces cell death of a cancer cell. Optionally, thecancer cell may be in vivo or in vitro. The cancer cell may be aprecancerous cell such as a myelodyplastic or myeloproliferative cell.In one embodiment, the cancer cell is a hematological cancer cell. Inone embodiment, the cancer cell is a leukemic cell, such as a cell froma subject with AML. In one embodiment, the DR receptor antagonist is aphenothiazine derivative such as thioridazine or chlorpromazine. In oneembodiment, the DR antagonist is selected from the compounds listed inTable 1.

As shown in Example 4 and FIG. 5, the Applicants have surprisingly shownthat some AML cell lines and primary AML cells exhibit a relativeincrease in the expression of dopamine receptors compared to normalhematopoietic stem cells. Screening subjects with cancer for theexpression of dopamine receptors in cancer cells may therefore serve toidentify subjects who would benefit from treatment with dopaminereceptor antagonists. Accordingly, in one aspect of the disclosure thereis provided a method for identifying a subject with cancer suitable fortreatment with dopamine receptor antagonists. In one embodiment, themethod comprises determining the expression of one or more dopaminereceptors in a sample of cancer cells from a subject. Subjects withcancer cells that express one or more dopamine receptors are therebyidentified as suitable for treatment with dopamine receptor antagonists.For example, the expression of one or more dopamine receptors in asample of cancer cells can be determined by testing the cancer cells forpolypeptides or polynucleotides that encode for dopamine receptors asdescribed herein. In one embodiment, the method includes obtaining orproviding a sample of cancer cells from the subject and/or testing thesample for the expression of one or more dopamine receptors. In oneembodiment, the cancer is leukemia and the cancer cells are leukemiccells. In one embodiment, the cancer is AML. Optionally, the methodincludes determining additional markers known to be associated withcancer, hematological malignancies, leukemia or AML or markersassociated with specific treatment regimes. In one embodiment, cancercells are also tested for the monocytic marker CD14.

The expression of dopamine receptors has been observed in samples ofbreast cancer and AML and can serve as a biomarker for the severity ofdisease. As shown in Example 11 and FIGS. 14 g-h, high levels of DRexpression correlate with poor prognosis while low levels demonstrate abetter prognosis. Accordingly, in one aspect of the disclosure there isprovided a method of determining a prognosis for a subject with cancer.In one embodiment, the method comprises determining the expression ofone or more biomarkers selected from dopamine receptor (DR) 1, DR2, DR3,DR4, DR5 and CD14 in a sample of cancer cells from the subject andcomparing the level of expression of the one or more biomarkers to acontrol. In one embodiment, an increase in the level of expression ofthe one or more biomarkers relative to the control indicates that thesubject has a more severe form of cancer. Optionally, the methodsdescribed herein include providing or obtaining a sample of cancer cellsfrom the subject such as a blood sample containing leukemic cells or atumour sample. In one embodiment, the cancer cells are leukemic cells orbreast cancer cells and increased expression of one or more biomarkerscompared to the control indicates a more severe form of leukemia orbreast cancer. Optionally, additional biomarkers known to be associatedwith cancer or severity of disease are also tested and compared tocontrol samples. A skilled person will appreciate selecting a controlthat is representative of a particular prognosis in subjects with cancersuch that observing a difference or similarity in the level of the oneor more of the biomarkers described herein between the test sample withthe control provides a corresponding prognosis for the test subject. Forexample, in one embodiment the control represents subjects diagnosedwith AML known to have a particular outcome or prognosis and observingan increase in the level of expression of one or more dopamine receptorsrelative to the control indicates a worse prognosis for the subjectrelative to the control.

The methods described herein are also useful for identifying subjectswith cancer. In one embodiment, the methods described herein are usefulfor identifying subjects with leukemia, such as AML or monocyticleukemia. Accordingly, in one embodiment, there is provided a method foridentifying a subject with leukemia comprising providing a sample from asubject and testing the sample for the expression of one or morebiomarkers selected from DR1, DR2, DR3, DR4 and DR5. In one embodiment,the sample comprises cancer cells such as leukemic cells and/or whiteblood cells. In one embodiment, the method comprises comparing the levelof expression of one or more biomarkers to a control. In one embodiment,increased expression of DRs in the sample compared to the control isindicative of cancer. In one embodiment, an increased expression of DRsin the subject compared to the control is indicative of leukemia. In oneembodiment, increased expression of DRs in the subject compared to thecontrol is indicative AML or monocytic leukemia. Optionally, the methodsdescribed herein may be used in combination with other diagnosticmethods for the identification of cancers or leukemia as known to aperson of skill in the art.

As shown in Example 11, dopamine receptor expression demarcates humancancer stem cells from other cells such as normal hPSCs that express thepluripotent marker SSEA3. Accordingly, in one embodiment, there isprovided a method of identifying a cancer stem cell from a population ofcells comprising determining whether a cell expresses one or morebiomarkers selected from dopamine receptor (DR) 1, DR2, DR3, DR4 andDR5. In one embodiment, expression of DR1, DR2, DR3, DR4 or DR5 isindicative that the cell is a cancer stem cell. Optionally, expressionof 2 or more, 3 or more, 4 or more or all 5 DRs is indicative that thecell is a cancer stem cell. In one embodiment, a cell that expressesDR1, DR2, DR3, DR4 and DR5 is identified as a cancer stem cell.

In one embodiment, the cancer stem cell is identified from a populationof cells. In one embodiment the population of cells contains more thanone cell type, such as somatic cells, pluripotent stem cells, cancercells and/or cancer stem cells. In one embodiment, the population ofcells is a plurality of cells in cell culture, such as tissue culture.In one embodiment, the population of cells is from a mammal, such as aprimary tissue sample or blood sample. In one embodiment, the populationof cells is from a mammal with cancer or suspected of having cancer. Inone embodiment, the population of cells includes stem cells, somaticstem cells and/or pluripotent stem cells as well as one or more cancerstem cells. In one embodiment, the population of cells includes cancercells or pre-cancerous cells such as hematological cancer cells. In oneembodiment, the population of cells includes monocytic cells. In oneembodiment, the population of cells includes breast cancer cells.Optionally, the population of cells is from a tissue sample, such as atumor sample, that has been dissociated into single cells.

In one aspect of the method, the step of determining whether the cellexpresses one or biomarkers comprises testing the cell for theexpression of polynucleotides or polypeptides that code for DR1, DR2,DR3, DR4 or DR5. For example, methods known in the art such a RT-PCR orreporter genes that detect the expression of polynucleotides, orimmunohistochemical methods that detect expression of polypeptides, canbe used for determining the expression of a biomarker such as DR1, DR2,DR3, DR4 or DR5. In one embodiment, the biomarkers are cell surfacebiomarkers and the method involves detecting DR1, DR2, DR3, DR4 or DR5expressed on the surface of the cell.

In one embodiment, the methods for identifying a cancer stem celldescribed herein include determining the level of expression of one ormore biomarkers selected from dopamine receptor (DR) 1, DR2, DR3, DR4and DR5 and then comparing the level of expression to a control level.For example, in one embodiment the control represents cells that are notcancer stem cells, such as somatic stem cells, hematopoietic stem cellsor cells that express the pluripotency marker SSEA3, and cells that havean increased level of expression of the biomarkers DR1, DR2, DR3, DR4and/or DR5 compared to the control are identified as cancer stem cells.Optionally, cells that have an increased amount of expression comparedto the control are identified as cancer stem cells (e.g. at least 2×,5×, or 10× etc.).

In one embodiment, the method can also comprise: (a) providing apopulation of cells (b) contacting the population with an agent thatspecifically binds to one or more biomarkers selected from DR1, DR2,DR3, DR4 and DR5; and (c) selecting cells that specifically bind to theagent of (b) thereby identifying and/or isolated cancer stem cells froma population of cells. In one embodiment, the agent is an antibody thatselectively binds to a biomarker. In one embodiment, the methodsdescribed herein can optionally include two or more selection orisolation steps. The methods described herein can also include anegative step selection, e.g., excluding cells that express one or moremarkers expressed in cells that are not cancer stem cells, or excludingcells that show reduced levels of expression of a particular marker.

In one embodiment, the present disclosure includes isolating cancer stemcells from a population of cells. For example, in one embodiment, cellsthat are identified as cancer stem cells are isolated from cells thatare not cancer stem cells or from other materials in a sample byselecting for or isolating cells that express one or more biomarkersselected from DR1, DR2, DR3, DR4 and DR5. Optionally, the cancer stemcells are isolated or selected using methods known in the art forsorting cells based on the expression of one or more biomarkers. Forexample, in one embodiment the step of isolating the cancer stem cellsform the population of cells comprises flow cytometry, fluorescenceactivated cell sorting, panning, affinity column separation, or magneticselection. In one embodiment, cells that express one or more dopaminereceptors are isolated using a binding agent that selectively bind todopamine receptors that is conjugated to a support such the matric in aseparation column or magnetic beads.

In one aspect of the disclosure, the methods described herein includedetermining the level of one or more biomarkers in a sample from asubject, such as the level of one or more dopamine receptors. In oneembodiment, the sample comprises cancer cells or is suspected ofcomprising cancer cells or pre-cancerous cells. For example, the samplecan comprise a blood sample, for example a peripheral blood sample, afractionated blood sample, a bone marrow sample, a biopsy, a frozentissue sample, a fresh tissue specimen, a cell sample, and/or a paraffinembedded section. In one embodiment, the subject has or is suspected ofhaving AML and the sample comprises mononuclear cells. In certainembodiments, the sample is processed prior to detecting the biomarkerlevel. For example, a sample may be fractionated (e.g. by centrifugationor using a column for size exclusion or by FACS using a biomarker formonocytes), concentrated or processed, depending on the method ofdetermining the level of biomarker employed.

The level of expression of the biomarkers described herein can bedetermined by methods commonly known to one of skill in the art. Forexample, in one embodiment, the level of one or more biomarkers isdetermined by measuring or detecting the level of a nucleic acid such asmRNA, or the level of a protein or polypeptide. In one embodiment,expression of the one or more biomarkers is determined by detecting thecell surface expression of DR1, DR2, DR3, DR4 and/or DR5. In oneembodiment, the methods described herein include detecting a biomarkerusing immunohistochemistry, such as by using an antibodies specific forthe biomarker or another biomarker-specific detection agent. Examples ofdopamine receptor antibodies suitable for use in the methods describedherein are also listed in Example 7 of the present disclosure.

In one embodiment, the level of an mRNA encoding for a biomarker isdetermined by quantitative PCR such as RT-PCR, serial analysis of geneexpression (SAGE), use of a microarray, digital molecular barcodingtechnology or Northern blot. A person skilled in the art will appreciatethat a number of methods can be used to determine the level of abiomarker, including mass spectrometry approaches, such as multiplereaction monitoring (MRM) and product-ion monitoring (PIM), and alsoincluding antibody based methods such as immunoassays such as Westernblots and enzyme-linked immunosorbant assay (ELISA). In certainembodiments, the step of determining the expression of a biomarker suchas one or more dopamine receptors as described herein, comprises usingimmunohistochemistry and/or an immunoassay. In certain embodiments, theimmunoassay is an ELISA. In yet a further embodiment, the ELISA is asandwich type ELISA.

The term “level” as used herein refers to an amount (e.g. relativeamount or concentration) of biomarker that is detectable or measurablein a sample. For example, the level can be a concentration such as μg/Lor a relative amount such as 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6,4.8, 5.0, 10, 15, 20, 25, 30, 40, 60, 80 and/or 100 times or greater acontrol level, standard or reference level. Optionally, a control is alevel such as the average or median level in a control sample. The levelof biomarker can be, for example, the level of protein, or of an mRNAencoding for the biomarker such as a dopamine receptor.

In one embodiment, when the level of two or more biomarkers isdetermined, the levels of the two or more biomarkers can be used togenerate an expression profile for the subject. For example, in oneembodiment, the methods described herein include determining a level fortwo or more biomarkers in the sample, generating an expression profilebased on the level of the two or more biomarkers and comparing theexpression profile to a control expression profile. A difference orsimilarity in the test sample expression profile and the controlexpression profile is then used to provide a prognosis for the testsubject, identify the subject as having cancer, or indicate whether thesubject is suitable for treatment with a dopamine receptor antagonist.

A further aspect of the disclosure includes the use of a dopaminereceptor antagonist for the treatment of cancer or a precancerousdisorder. In a similar aspect, there is provided a dopamine receptorantagonist for use in the treatment of cancer or a precancerousdisorder. In one embodiment the cancer is leukemia. In one embodiment,the leukemia is acute myeloid leukemia or monocytic leukemia. In oneembodiment, the dopamine receptor antagonist is a phenothiazinederivative such thioridazine or chlorpromazine. In one embodiment, theDR antagonist is selected from the compounds listed in Table 1.

Also disclosed herein is the use of a dopamine receptor antagonist forthe manufacture of a medicament for the treatment of a cancer and/or aprecancerous disorder.

A further aspect of the disclosure includes methods of screeningcompounds for anti-cancer activity comprising identifying compounds thatantagonize one or more dopamine receptors. In one embodiment, compoundsthat antagonize dopamine receptors are identified as having anti-canceractivity. In one embodiment, the methods include screening compounds toidentify those that reduce the proliferation of cancer stem cellsrelative to normal stem cells such as hematopoietic stem cells as setout in Examples 7 and 8 of the present description.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1 Thioridazine is Cytotoxic to Leukemic Cell Lines

The effect of Thioridazine on normalized cell number was evaluated in 3leukemic cells lines: HL-60, MV4-11 and OCI-AML3. All three lines areleukemic cell lines. HL-60 was derived from promyelocytic AML whereas MV4-11 and OCI-AML3 are representative of AML. Each compound was incubatedwith the cells for 72 h. The control was DMSO (ie the vehicle used forthe compound) for 72 h. Each condition had three replicates.

As shown in FIG. 1, doses of 0.1 μM and 1 μM thioridazine had littleeffect on normalized cell number, while at 10 μM the normalized cellnumber was reduced to almost zero.

Example 2 Differential Activity of Thioridazine on AML Blast-FormingPotential and Colony Forming Potential of Normal Stem Cells

The effects of thioridazine on blast formation in an AML cell line wascompared to the effect of thioridazine on colony formation in normalhuman stem cells.

Normal HSCs and progenitors were sourced from either mobilizedperipheral blood or umbilical cord blood of health patients. Primary AMLcells were taken from patients diagnosed with AML. Both normal HSCs andprimary AML cells were cultured under standard in vitro methocelluloseassay conditions (seehttp://www.stemcell.com/en/Products/All-Products/MethoCult-H4434-Classic.aspxas well as Clinton Campbell et al. The human stem cell hierarchy isdefined by a functional dependence on Mcl-1 for self-renewal capacity.Blood 116 (9) 1433-1442 (Jun. 4, 2010), hereby incorporated byreference) for at least 14 days before the number of colonies wererecorded. As shown in FIG. 2, 10 μM thioridazine has a differentialeffect on normal HSCs versus AML cells. 10 μM thioridazine reduced thecolony forming potential of normal HSCs from about 100 (CTRL treatedwith DMSO) to about 66 total colonies (FIG. 2A), but had a much moresignificant effect on AML cells reducing the number of CFU colonies toabout 22 blast colonies (FIG. 2B) to 1.6 blast colonies.

FIG. 3 shows cell pellets of CFU colonies generated from normal HSC andAML treated with thioridazine. At a dose of 10 μM, pelleted cells arestill visible for HSCs, but not for AML cells. Thioridazine thereforeselectively targets Blast-CFU Potential of AML cells.

Example 3 Chlorpromazine is Toxic to AML Cell Lines

The dopamine receptor antagonist and phenothiazine-related compoundchlorpromazine was also investigated for effects on the AML cell linesHL-60, MV4-11 and OCI-AML3. Testing was performed as set out inExample 1. As shown in FIG. 4, 10 μM Chlorpromazine is toxic to AML celllines.

Example 4 Expression of Dopamine Receptors in Normal Blood VersusLeukemia

The expression of the dopamine receptors DR1, DR2, DR3, DR4 and DR5 wereanalyzed in AML cell lines HL-60, MV4-11, AML-OCI2 and AML-OCI3),Primary AML cells (AML22101, AML29428, AML22174, AML29560) isolated fromAML patients, normal blood mononuclear cells (MNC) (MPB21471 andMPB28137; healthy patient blood) as well as umbilical cord blood primarycells enriched for normal Human Stem Cells or progenitors (CB107, CB108and CB109) using StemSep® Human Hematopoieitc Progenitor Cell enrichmentkit(http://www.stemcell.com/en/Products/All-Products/StemSep-Human-Hematopoietic-Progenitor-Cell-Enrichment-Kit.aspx)and enrichment levels of HSCs/Human Progenitor cells confirmed by flowcytometry. Isotype expression was measured as background. Peaks to theright of the isotype peak represent positive expression of DR markers.

As shown in FIG. 5, dopamine receptors are expressed on primary AML, AMLcell lines and normal mononuclear blood cells (MNC) but not in bloodenriched for normal HSCs (CB(lin−). The data shows that when the sampleis positive for DR expression that all five DR subtypes are usuallypresent.

Not all primary AMLs were observed to express dopamine receptors.Accordingly, subjects may be pre-screened for the expression of dopaminereceptors in order to identify subjects suitable for AML treatment withDR antagonists. Optionally, pre-screening of subjects may encompass allfive DR subtypes, or specific subtypes or combination of subtypes.

Example 5 Multiple DR Antagonists are Cytotoxic to AML Cell Lines

A series of dopamine receptor agonists, D₃₋antagonists,DR_(1 & 5-)antagonists and multi-receptor antagonists were tested forcytotoxicity against three AML cell lines HL-60, OCI-AML2 and OCI-AML3.Testing was performed as set out in Example 1.

As shown in FIG. 6, CLOZ at higher concentrations as well as CHL andTHIO have a significant effect on cytotoxicity of AML cell lines.Without being limited by theory, the cytotoxic effect may requireinhibition of multiple dopamine receptors. THIO, CHL and CLOZ beingmultireceptor antagonists work to eradicate the AML cell lines while theD₃ and DR_(1 & 5)-specific antagonists only reduce cell count to 60%.

Example 6 Dopamine Receptors are Expressed in the CD14+ Cell Populationof Primary AML

The expression of dopamine receptor subtypes was analyzed in primary AMLcells. Primary AML cells obtained from AML patients were co-stained withantibodies specific to the DR subtype and CD14 prior to being analyzedusing flow cytometry. The majority of DR+ cells were found to bepositive for CD14.

As shown in FIG. 7, the expression of the CD14 monocytic marker iscorrelated with the expression of each DR subtype.

The effects of thioridazine were also examined on a subpopulation ofCD14+ cells in primary AML. Primary AML cells were cultured undercontrol (DMSO vehicle) or 10 uM thioridazine for 72 h and then stainedfor with antibodies specific to CD14. The number of CD14+ cells in bothcontrol and thioridazine treated samples was determined using flowcytometry and the frequency of CD14+ cells was found to be lower in thethioridazine treated sample, suggesting that this compound selectivelytargets the CD14+ subpopulation in AML cells.

As shown in FIG. 8, 10 μM thioridazine also reduced the normalizedfrequency of CD14+ cells in primary AML cells, showing that thioridazineselectively targets CD14+ cells. The AML control group contained afraction of CD14+ cells. This fraction is reduced with thioridazinetreatment and is represented as a reduction in the normalized frequencyof the control (100%) versus treated (20%).

Example 7 Identification and Characterization of Drugs that InduceDifferentiation of hPSCs

Identification of drugs that target cancer stem cells (CSCs) withoutaffecting normal stem cells (SCs) would be ideal for future cancertherapies, but is limited by the lack of assays for both CSCs and normalSCs in the human that are amenable to robust biological screens. As setout in the following examples, using a neoplastic vs. normal humanpluripotent stem cell (hPSC) differentiation platform, compounds wereidentified that are not toxic, but induce differentiation to overcomeneoplastic self-renewal of CSCs. Of the several candidate anti-CSCagents identified, thioridazine, an approved anti-psychotic drug, wasable to selectively target human somatic CSCs capable of in vivoleukemic disease initiation while having no effect on normal blood SCcapacity. Antagonism of dopamine receptor (DR) signaling by thioridazineforms the basis of selective CSC targeting, and revealed DR as abiomarker for CSCs of hematopoietic and breast tumor origins.

Experimental Procedures

Generation of Neoplastic hPSC EOS-GFP Lines.

Neoplastic v1H9 or v2H9 hPSC cells (Werbowetski-Ogilvie et al., 2009)were transduced with lentivirus bearing the EOS-C3+ or EOS-S4+ vectorsprovided by Dr James Ellis (Hotta et al., 2009). After lentiviraltransduction cells were selected using Puromycin, and subsequentlysorted as single cells into a 96-well plate based on GFP expressionusing a FASCAria II (Becton-Dickinson). Colonies generated from singlecell clones were used to establish the v1H9-Oct4-GFP (EOS-C3+),v2H9-Oct4-GFP (EOS-C3+) and v1H9-Sox2-GFP (EOS-S4+) lines.

Cell Culture.

The H9 hESC, v1H9, v1H9-Oct4-GFP, v2H9-Oct4-GFP, v1H9-Sox2-GFP andfibroblast-derived iPSCs were cultured as previously described (Chadwicket al., 2003; Werbowetski-Ogilvie et al., 2009).

Primary Human Samples.

For AML specimens, peripheral blood and/or bone marrow was collected atthe time of clinical presentation. Healthy hematopoietic cells wereobtained from umbilical cord blood samples. All samples were obtainedfollowing informed consent according to Research Ethics Board approvedprotocols at McMaster University and the London Health Sciences Centre.Human breast tumor samples were obtained from reduction mammoplastysurgeries following informed consent according to Research Ethics Boardapproved protocols at McMaster University.

In Vitro Culture Platform for Normal and Neoplastic hPSCs.

Chemical screens involved v1H9-Oct4-GFP cells seeded at 5,000 cells perwell in mouse embryonic fibroblast conditioned media (MEFCM)supplemented with 8 ng/ml bFGF. 24 hours later the media was exchangedfor MEFCM with compounds at 10 μM and 0.1% DMSO, 0.1% DMSO (−BMP4) or100 ng/ml of BMP4 and 0.1% DMSO (+BMP4) for 48 hours before beingexchanged with fresh media with compound for a further 24 h (totalcompound treatment time 72 h) prior to being fixed and prepared forautomated imaging and plate reader analysis. Confluent H9 &fibroblast-derived iPSC were seeded at 10,000 cells per well in MEFCMsupplemented with 8 ng/ml bFGF. 24 hours later the cells were treatedwith compounds at 10 μM and 0.1% DMSO, 0.1% DMSO (−BMP4) or 100 ng/ml ofBMP4 and 0.1% DMSO (+BMP4). Fresh MEFCM supplemented with compounds wasexchanged daily for 5 days. On day 5, hPSC's were fixed and prepared forautomated imaging and plate reader analysis. See supplementaryexperimental procedures for further details.

Teratoma Assay.

400,000 H9 hESCs or v1H9-Oct4-GFP were injected intra-testicularly intomale NOD/SCID mice and teratomas analyzed for Oct4 as previouslydescribed. (Werbowetski-Ogilvie et al., 2009).

Xenotransplantation Assays.

NOD.Cg-Prkdc^(scid) II2rg^(tm1Wjl)/SzJ adult mice (NSG) weresub-lethally irradiated with 315 rads 24 hours prior transplantation.0.8−1.0×10⁷ AML MNCs or 1.5−1.8×10⁵ CB lin− hematopoietic cells treatedwith compound or DMSO-vehicle for 24 h were injected via tail vein (IV).After 6-10 weeks, animals were culled, and the BM and spleen wereanalyzed for the presence of human cells by flow cytometry (LSRII, BD)and data was analyzed using FlowJo software (Tree Star Inc). Forsecondary HSPC transplants, equal number of engrafted human cells fromCB lin− transplants were injected IV in adult irradiated NSG mice asdescribed for primary transplants.

Statistical Analysis.

Data is represented as the mean±SEM or mean±SD. Significant differencesbetween groups were determined using unpaired two-way or one-wayStudents' t test.

Pluripotent Stem Cell Culture.

The H9 hESC, v1H9, v1H9-Oct4-GFP, v2H9-Oct4-GFP, v1H9-Sox2-GFP andfibroblast-derived iPSCs were cultured on Matrigel™-coated (BDBiosciences 353234) plates with mouse embryonic fibroblast-conditioned(MEFCM) media supplemented with 8 ng/ml bFGF (GIBCO 13256-029). MEFCM iscomposed of KO-DMEM (GIBCO 10829-018), 20% KO-Serum Replacement (GIBCO10828-028), 1% Non-Essential Amino Acids (GIBCO 11140-050), 1 mML-Glutamine, 0.1 mM β-mercaptoethanol (Sigma Aldrich M7522). Cell lineswere passaged every 7 days using 100 Units/mL of Collagenase IV (GIBCO17104-019) for 2-3 minutes. Cell seeding density, assay duration andDMSO vehicle concentration in 96 wells were optimized for v1H9-Oct4-GFPcells and normal H9 hPSC. For v1H9-Oct4-GFP, an optimum initial seedingdensity of 5,000 cells per well for 72 h of treatment was selected basedon maximal levels of GFP and z′ discrimination between ±BMP4 controls.For normal hPSC, an optimal seeding density of 10,000 cells per well wasselected based on maximal z′-prime discrimination between ±BMP4controls.

Primary Human Samples.

Mononuclear cells were prepared using Ficoll-Paque Premium (GEHealthcare). For hematopoietic cells, lineage depletion was performedusing EasySep (StemCell Technologies) following manufacturer'srecommendations.

AML/HPSC Cell Culture.

AML cell lines, namely, OCI-AML2 (M4), OCI-AML3 (M4), HL-60 (M2) andMV-4-11(M5) were cultured in RPMI (Gibco) supplemented with 5%heated-inactivated FBS (HyClone). For DR agonist studies withR(+)-7-Hydroxy-DPAT hydrobromide (Sigma), serum-free conditions wereemployed instead due to the prevalence of dopamine in FBS (Little etal., 2002). AML patient blasts were cultured in IMDM supplemented with5% heated inactivated FBS (HyClone), 5 ng/mL IL3 (R&D systems), 5×10⁻⁵ Mβ-mercaptoethanol (Sigma) and BIT (StemCell Technologies). HSC mediacontained IMDM supplemented with 1% BSA (Sigma), 100 ng/mL SCF (R&Dsystems), 100 ng/mL Flt-3L (R&D systems) and 20 ng/mL TPO (R&D systems).Patient HSPC and AML samples were treated with compound or DMSO-vehicle(0.1%) for 24 h prior to CFU plating or xenotransplantation studies.

Antibodies.

Antibodies used for immunocytochemistry were the following: Oct3/4 (BDTrunsduction Laboratories, cat#611203), Sox2 (R&D, cat#AF2018). Todetect human hematopoietic cells, Pacific Blue-, PE-, APC- or FITClabeled anti-human CD45 was used (BD Biosciences). FITC anti-CD33, PEanti-CD13, FITC anti-CD41a, FITC anti-HLA DR, and PE anti-CD19antibodies were obtained from BD Pharmingen. PE anti-CD14, PE anti-CD15and PE anti-GlyA were acquired from Immunotech Beckman Coulter. Todetermine pluripotency, PE anti-SSEA3 (BD Biosciences) and PE- orAlexaFluor488 anti-Oct4 (BD Biosciences). Rabbit anti-human dopaminereceptor antibodies; DRD1 (Cat#324390), DRD2 (Cat#324393), DRD3(Cat#324402), DRD4 (Cat#324405) and DRD5 (Cat#324408) were sourced fromEMD Chemical. Anti-rabbit Alexa-Fluor-488 (Molecular Probes) was used asthe secondary antibody. Primary anti-p53 (Cat#2527) and anti-p21(Cat#2947) rabbit IgG sourced from Cell Signaling Technology were usedto stain fixed and permeabilized cells. Anti-rabbit alexa-Fluor-546(Molecular Probes) was used as the secondary antibody. For breast tumorstaining, APC anti-CD44 and PE-CD24 were sourced from BD Pharmingen.

Automated Imaging and Analysis

Imaging Neoplastic hPSC.

Cells were fixed in 2% paraformaldehyde and stained with 10 μg/mLHoechst 33342 (Invitrogen) with a Combi Multidrop Dispenser (Thermo).For experiments that involved Oct4 immunocytochemistry, a monoclonalantibody for Oct4 (BD) was used along with an Alexa-Fluor-647 secondary(Invitrogen). Immunocytochemical staining was performed by a Janusautomated liquid handler (Perkin Elmer). Images were acquired at 10×N.Awith an Arrayscan HCS VTI Reader (Cellomics) by means ofepi-fluorescence illumination and standard filter sets.

Imaging Normal hPSC.

Cells were fixed in 2% paraformaldehyde and stained with 10 μg/mLHoechst 33342 (Invitrogen). Standard fluorescence immunocytochemicaltechniques were used to stain the cells with a monoclonal antibody forOct4 (BD), and an Alexa-Fluor-647 secondary antibody (Invitrogen). Allsteps were performed by a Janus automated liquid handler (Perkin Elmer).Images were acquired at 5× with an Arrayscan HCS Reader (Cellomics) bymeans of epi-fluorescence illumination and standard filter sets.

Image Analysis.

Image analysis was performed using custom scripts in Acapella software(Perkin Elmer). Nuclear objects were segmented from the Hoechst signal.For neoplastic cell lines, object intensity analysis was performed onGFP positive cells only. For normal cell lines, the fraction ofAlexa-Fluor-647-positive cells was quantified. Images and well-leveldata were stored and analysed in a Columbus Database (Perkin Elmer) andfurther data analysis, compounds registration and hit identification inActivityBase (IDBS).

Gene Expression Analysis.

Cells in specific conditions were collected and RNA was extracted byusing RNeasy kit (Qiagen), complementary DNA (cDNA) generation by usingSuperScript III® cDNA synthesis kit (Invitrogen), pre-amplification andTaqMan® array reaction (Applied Biosystems) were performed according tomanufacturer's instructions. The gene expression profile for eachtreated cell population was analyzed using TaqMan® Stem CellPluripotency Array Card on ViiA 7 Real-Time PCR System (AppliedBiosystems). Each reaction sample was dispensed into loading wells onthe array card and centrifuged twice at 336×g for 1 min each time,sealed, and placed in the thermal cycler. The following cyclingconditions were used for all array card applications: 45° C. for 10 min,94° C. for 10 min, and 40 cycles of 94° C. for 30 s followed by 60° C.for min. Array data were normalized to 18S RNA and GAPDH and comparisonswere performed using data analysis 2.0 software (Applied Biosystems).

Methylcellulose Colony-Forming Assay.

AML patient or CB lin− cells were cultured 24 hours in the presence ofcompound or DMSO-vehicle (0.1%) control. AML cells were plated at 50 000cells/mL in Methocult GF H4434 (Stem Cell Technologies). CB lin− cellswere plated at 1000 cells/mL in Methocult GF H4434 (Stem CellTechnologies). Colonies were scored after 14 days of culture usingstandard morphological criteria.

Volumetric Cell Counting.

The number of AML-OCI2 and AML-OCI3 cells present after 72 h treatmentwith DR antagonists (FIG. 16 b) and agonist (FIG. 16 c-d) were countedby measuring the number of events within a fixed volume following thegrating strategy defined by forward scatter and side scatter clustering,7AAD− and Hoechst+.

Human Breast Cancer Sample Processing.

Human breast tumor samples were obtained from reduction mammoplastysurgeries following informed consent according to Research Ethics Boardapproved protocols at McMaster University. The breast tumor chunks werecut into small fragments (chunks of less than 1 mm) with scissors andscalpel. Subsequently, 3 mL of Versene (1 mL of 0.5M EDTA in 1 L of1×PBS) and 7 mL of trypsin-collagenase solution were added for each gramof tumor tissue and incubated for 30 min at 37° C. Thetrypsin-collagenase solution consisted of RPMI 1640 (Gibco #11875093),2% penicillin/streptomycin (Invitrogen #15140163), 1% FungizoneAntimycotic (Invitrogen #15290018), 2% FBS, 3 mg/mL Collagenase A (RocheDiagnostics #11088793001), and 0.1% of 2.5% trypsin (Gibco #15090). Anequal volume of RPMI 1640 with 2% FBS was then added to the tissuesuspension. The tissue suspension was filtered through a 40 μm nylonstrainer (Falcon #352340). The supernatant was discarded and the cellpellet was resuspended in 10 mL of F-12+GlutaMAX Nutrient Mixture Ham 1×(Gibco #31765) supplemented with 2% penicillin/streptomycin and 1%Fungizone Antimycotic. Viable cells were counted using a hemocytometerand Trypan blue solution and prepared for flow cytometry. Antibodystaining included Rabbit anti-human dopamine receptor antibodies; DRD1(Cat#324390), DRD2 (Cat#324393), DRD3 (Cat#324402), DRD4 (Cat#324405)and DRD5 (Cat#324408) were sourced from EMD Chemical and Anti-rabbitAlexa-Fluor-488 (Molecular Probes) was used as the secondary antibodyalong with APC anti-CD44 and PE-CD24, both sourced from BD Pharmingen.

Example 8 High Throughput Screening Identification of Compounds thatInduce Differentiation of Neoplastic hPSCs

The inventors have previously described a variant human pluripotent stemcell (hPSC) line that displays neoplastic features which includeenhanced self-renewal and survival, along with aberrant block interminal differentiation capacity in vitro and in vivo(Werbowetski-Ogilvie et al., 2009). Based on these similarities infunctional properties to somatic CSCs, neoplastic hPSCs were examined asa surrogate for somatic CSCs that would be amenable for high content andhigh throughput screening in vitro. A screening platform was developedto identify small molecules that selectively target neoplastic hPSCswhilst having little effect on normal hPSCs. This differential screeningplatform is capable of identifying potent candidate drugs thatselectively target somatic CSCs while sparing healthy SC capacity.

Oct4 and Sox2 provide a reliable indicator of loss of self-renewingpluripotent state and differentiation induction of normal and neoplastichPSCs. To provide a more straightforward method for detecting loss ofOct4 or Sox2 during induced differentiation of neoplastic hPSCs,GFP-reporter lines were generated by transduction of neoplastic hPSCswith the EOS-GFP reporter (v1H9-Oct4-GFP and v1H9-Sox2-GFP,respectively) (Hotta et al., 2009). GFP intensity was observed to becorrelated with Oct4 and Sox2 expression in treatments that favoredself-renewal stability and conditions that induce differentiation withthe addition of BMP4. This response was consistently found using anadditional neoplastic hPSC line, v2H9 (Werbowetski-Ogilvie et al., 2009)transduced with the same EOSlentivirus GFP-reporter (v2H9-Oct4-GFP), aswell as a Sox2 reporter line (v1H9-Sox2-GFP).

The uniform response to differentiation and maintenance of pluripotencyin all hPSC cell lines generated also revealed that viral integration orclonal selection by EOS reporter construct insertion is irrelevant toresponsiveness. These results suggest that compounds that inducedifferentiation can be identified based on the reduction of GFPintensity in neoplastic hPSC reporter lines and could be exploited forchemical screening. To that end, conditions for automated high contentmicroscopy and fluorimetric-based high throughput screening were used todetect reductions in pluripotency marker expression of hPSCs.Microscopic analysis of normal hPSCs showed that distinct Oct4+ cellsare lost following BMP4 treatment. Similarly, the reduction in both GFPand Oct4 due to BMP4 treatment of neoplastic Oct4-GFP hPSCs wasquantified by high content microscopy and plate reader-basedfluorimetry. To identify ideal candidates for targeting CSCsdifferentiation of both normal and neoplastic hPSCs in response tocompound treatment was assessed in parallel.

Given the validation of the screening platform a chemical librariescomposed of 590 well-established annotated compounds from the NIHClinical Collection and Canadian Compound Collection was screened. TheseCollections have been previously scrutinized in numerous other mammaliancell lines (Diallo et al., 2010; Shoemaker, 2006). Following thedemonstration that fluorometric highthroughput screening (HTS) and highcontent screening (HCS) platforms give equivalent measurements for lossof pluripotency (GFP RFU and mean GFP intensity per cell, respectively)and cell count (Hoechst RFU and Cell count, respectively) of the 51defined compounds, HTS was selected as the preferred platform for morerapidly screening compound libraries (FIG. 9 a). Of the 590 compoundsscreened (at 10 μM based on previous studies (Inglese et al., 2007)), 11compounds were identified to induce differentiation as indicated by areduction in both GFP residual activity (% RA) and Hoechst % RA (FIGS. 9b-c). A total of 4 of these compounds; indatraline, thioridazine,azathioprine, and mefloquine, were identified as candidate compoundsbased on clustering and levels of Hoechst % RA in excess of 30% (FIG. 9b). Secondary high content analysis revealed indatraline to be aquestionable candidate and was thus excluded, whereas content analysisand HTS analyses dually confirmed thioridazine, azathioprine, andmefloquine as candidate compounds (FIG. 9 d) and were thus selected forfurther testing (FIGS. 9 e-g). When compared to control-treated hPSCs,each compound appeared to induce distinct morphological changes inneoplastic hPSCs (FIG. 9 e). Reduction in GFP intensity was confirmedusing image analysis (FIG. 9 f) and further assessed over a wide rangeof doses to calculate half-maximal effective concentration (EC50) foreach compound (FIG. 9 g). Only thioridazine and mefloquine were found topossess EC50 values lower than the 10 μM target threshold (FIG. 9 g) andthus defined as candidates for further in depth evaluation usingneoplastic hPSCs and somatic CSCs from patients.

Example 9 Thioridazine Selectively Induces Neoplastic hPSCDifferentiation and Reduces Human AML Blasts without Affecting NormalHematopoietic Stem/Progenitor Cells

The responses to thioridazine and mefloquine were evaluated in bothnormal (FIG. 10 a) and neoplastic hPSCs (FIG. 10 b) at threeconcentrations using quantitative flow cytometry to detect the loss ofOct4 and reveal the degree of differentiation. Salinomycin, a reportedselective inhibitor of breast CSCs (Gupta et al., 2009), was includedfor comparison. At 10 μM, all compounds reduced the number of cells, butthe levels of Oct4 in remaining normal hPSCs was not below levelsobserved with BMP4 treatment (FIG. 10 a). This same response wasreplicated in fibroblast-derived human iPS cells, (FIG. 11 a),representing an additional normal hPSC line from a distinct (adult)origin, indicating the effects are not specific to embryonic sources.When the same compounds were used to treat neoplastic hPSCs, mefloquineand thioridazine treatments caused reductions in cell number and thelevels of Oct4 in neoplastic hPSCs. Only thioridazine was able to reducelevels of Oct4 below BMP4 differentiation controls (FIG. 10 b),indicating the ability of thioridazine to overcome neoplastic hPSCdifferentiation block. A more comprehensive dose response of allcompounds was performed on neoplastic hPSCs to confirm this response(FIG. 11 b). To identify compounds that selectively differentiateneoplastic hPSCs quantitatively, the ratio of normalized percentage ofOct4+ cells between normal and neoplastic hPSCs in response to thesecompounds was determined. For example, a ratio of 1 suggests equivalentdifferentiation whereas a ratio>1 defines relatively moredifferentiation in neoplastic hPSCs vs. normal hPSCs. Only thioridazine,at both 1 μM and 10 μM, had a significant impact on inducingdifferentiation of neoplastic hPSCs over normal hPSCs (FIG. 10 c). Rapidaccumulation of the cell stress marker p53 (FIG. 10 d) and itstranscriptional target p21 (FIG. 10 e) were used to further distinguishdifferentiation induction from cellular toxicity. Treatment ofneoplastic hPSCs with the toxic chemotherapeutic agent etoposideresulted in high levels of p53 and p21 after 24 h. However, treatmentwith 10 μM thioridazine or BMP4, unlike agents that induce toxicityalone, resulted in no accumulation of p53 or p21, consistent withinduced differentiation rather than stress-response programs.Furthermore, thioridazine treatment led to expression of differentiationgenes quantified by TaqMan Low-Density Array-qPCR in neoplastic hPSCs.An upregulation in 21 of 50 differentiation-associated genes (FIG. 10 f)was observed in treated neoplastic hPSCs consistent withdifferentiation-inducing effects of thioridazine.

To examine the potential similarities in chemical response of neoplastichPSCs to somatic CSCs, normal and neoplastic populations of the humanhematopoietic system were assessed. Experimentally, self-renewal anddifferentiation of both human hematopoietic stem-progenitor cells(HSPCs) and Leukemic Stem Cells (LSCs) can be interrogated by powerfuland well established in vitro and in vivo assays uniquely available tothe hematopoietic system, making it an ideal tissue to evaluate thepotential surrogacy of using normal and neoplastic hPSCs as a primaryscreening tool for anti-CSC compounds. Lineage-depleted umbilical cordblood (CB lin−) is highly enriched for HSPCs and is a reliable source ofnormal somatic SCs capable of self-renewal and multilineagedifferentiation to all blood lineages. Acute myeloid leukemia (AML) is ahematological neoplasia characterized by a block in mature myeloiddifferentiation that is sustained by a self-renewing LSC (Bonnet andDick, 1997; Lapidot et al., 1994).

As such, progenitor assays in methylcellulose were conducted with HSPCsand 5 AML patient samples; each treated with thioridazine, mefloquine,or salinomycin in order to assess each compound's impact on in vitroclonogenic and multilineage hematopoietic differentiation.Representative cell pellets of the total colony-forming units (CFUs)generated from HSPCs (FIG. 10 g) and AML (FIG. 10 h) treated with eachcompound are shown. Thioridazine treatment resulted in a reduction inAML proliferation/clonogenic capacity while retaining HSPC multilineagedifferentiation (FIG. 11 c). Changes in multilineage differentiationwere quantified based on the enumeration of CFUs generated followingtreatment of HSPCs (FIG. 10 i) and AML patient (FIG. 10 j) samples withthese compounds. At both 1 μM and 10 μM salinomycin reduced AML-blastCFU potential (FIG. 10 j), but also reduced HSPC CFU potential over alldoses tested (FIG. 10 i) indicative of non-specific toxicity in thehematopoietic system. In contrast, mefloquine and thioridazine reducedAML-blast CFU formation (FIG. 10 j) while having little effect on HSPCCFU potential (FIG. 10 i) and multilineage composition (FIG. 11 d)indicating that mefloquine and thioridazine do not alter normalhematopoiesis.

The most desired outcome of compounds identified toward clinical usewould entail preferential elimination of AML-blast CFU generation whilepreserving normal HSPC progenitor capacity. The ratio between total CFUsgenerated from HSPC vs. AML-blasts to reveal the highest selectivity fortargeting AML was calculated (FIG. 10 k). A ratio of 1 suggestsequivalent normal to neoplastic progenitor potential whereas a ratio>1defines a compound that selectively reduces AML-blast CFU potential.Salinomycin (1 μM), mefloquine (10 μM), and thioridazine (10 μM) dosesyielded the highest ratio values for each compound (FIG. 10 k) and werethus selected for in vivo evaluation. Thioridazine 10 μM, in particular,demonstrated the highest ratio of all compounds, but most importantlywas the only compound to show a significantly lower AML-blast CFUpotential relative to normal HSPC CFU potential (FIG. 10 k). To addresswhether thioridazine's specificity for reducing the clonogenic potentialof AML-blast CFUs was due to induction of differentiation, the frequencyof CD11b, a marker of granulocytic maturation, in patient AML cells wasassayed in response to thioridazine treatment (FIG. 10I). A markedincrease in the frequency of granulocytic AML-blast cells was observedwith treatment duration (FIG. 10I) indicating that thioridazine exhibitsits specific targeting of AML cells through induction ofdifferentiation. This finding is analogous to differentiation-inductiondemonstrated in neoplastic hPSCs (FIG. 10 a-f) and confirms the robustreadout of this screening platform towards identifying agents able todifferentiate neoplastic cells. This result also suggests thatthioridazine may represent the best candidate for specific targeting ofAML CSCs that requires testing using in vivo human-mouse xenograftassays.

Example 10 Thioridazine Reduces LSC Function while Sparing Normal HSPCs

To delineate whether the inhibition of AML-blasts detected in vitro wasdue to the compounds affecting the neoplastic stem cell compartment,xenotransplantation studies (Dick, 2008) that functionally define LSCsand hematopoietic stem cells (HSCs) were conducted (FIG. 12). Treatmentof HSPCs with salinomycin (1 μM) significantly reduced hematopoieticengraftment to almost non-detectable levels (FIG. 13 a) revealing thatthis compound interferes with normal hematopoiesis from HSPCs and wasthus excluded from further evaluation as it is unlikely to provide theselective anti-CSC therapeutic targeting desired. In contrast,mefloquine (10 μM) treatment displayed a slight, yet insignificant,reduction in HSC capacity relative to controls (FIG. 12 a). However,mefloquine proved ineffective in reducing AML LSC capacity and was thusdiscontinued from further evaluation due to absence of selective effects(FIG. 12 c).

In contrast to both salinomycin and mefloquine, treatment of HSPCs withthioridazine 10 μM displayed the same level of bone marrow (BM)engraftment (FIG. 12 a) and splenic engraftment (FIG. 13 b) as controlvehicle treated cells. Multilineage reconstitution capacity wasidentical from control- and thioridazine-treated human HSCs with myeloid(FIG. 12 b), lymphoid (FIG. 12 b), erythroid (FIG. 13 d), andmegakaryocytic development (FIG. 13 d) completely unaffected. Asmeasured by secondary serial transplantation, thioridazine treatment didnot affect HSC self-renewal as compared to control-treated samples (FIG.13 f). However, in sharp contrast to salinomycin and mefloquine,thioridazine treatment was able to significantly reduce leukemicdisease-initiating AML LSCs (FIGS. 12 c-d; FIG. 13 c; FIG. 13 e).Calculating the ratio of HSPC normal hemaotopoietic regeneration (%hCD45+) to AML leukemogenesis (% CD33+hCD45+ blasts) revealed thatthioridazine significantly reduced LSC function while preserving normalHSC capacity (FIG. 12 e). In the absence of thioridazine, no differencein the level of leukemic engraftment of secondary transplant recipientswas observed. This suggests that continued exposure to this drug isnecessary to inhibit leukemogenesis in secondary recipients. These datademonstrate that thioridazine selectively targets somatic CSCs whilsthaving no effect on normal SC properties in vivo. As thioridazine wasidentified through the use of a novel differential screening platformusing normal and neoplastic hPSCs in vitro, the functional effects ofthioridazine provide an example of the predictive value of using humanPSCs to understand somatic CSCs.

Example 11 Dopamine Receptors Demarcate Human CSCs

Thioridazine is known to act through the dopamine receptors (DR 1-5)(Beaulieu and Gainetdinov, 2011; Seeman and Lee, 1975). To assesswhether the mechanism of thioridazine action to selectively interferewith human CSCs vs. normal SCs is via DR antagonism, DR cell surfaceexpression was analyzed. To date, five DRs have been identified anddivided into D₁-family (D1 and D5) and D₂-family (D2, D3, and D4)receptors (Sibley and Monsma, 1992). Normal hPSCs expressing thepluripotent marker SSEA3 were devoid of DR expression (FIG. 14 a andFIG. 15 a-b). In contrast, neoplastic hPSCs expressed all five DRs (FIG.14 b). The observed differential expression of DRs and the selectiveinhibition of thioridazine for neoplastic hPSCs suggest that inhibitionof DR signaling may play a role in selective targeting of human CSCs vs.normal SCs.

To expand the potential role of DRs in CSCs based on the functional roleof thioridazine treatment we examined whether DR antagonism couldaccount for the loss of LSC function following thioridazine treatment.Expression of DR1-5 was analyzed in HSPCs (FIG. 14 c) and humanhematopoietic mononuclear cells from normal CB (FIGS. 15 c-f) and AMLpatient samples (FIG. 14 d and FIG. 15 g). DRs were not observed in theprimitive HSCs or progenitor populations of CB (identified as theCD34+38− or CD34+38+ fractions, respectively (Bhatia et al., 1997))(FIG. 14 c) indicating that HSCs and progenitors do not express thetargets for thioridazine. Similarly, DRs were undetectable on thesurface of erythroid (FIG. 15 c), megakaryocytic (FIG. 15 c), andlymphoid cells (FIG. 15 d). Only monocytes defined as CD14+ andapproximately half the population of granulocytes defined as CD15+expressed DRs (FIGS. 15 e-f). All of the 13 AML patient samples analyzedcontained a population of DR+ blasts with varying levels of all fivereceptors (FIG. 14 d) and were predominately detected in CD34+/CD14+cells (FIG. 15 g). However, unlike normal HSCs, CD34+ cells do notcorrelate with LSC capacity in human AML (Taussig et al., 2008) and haverecently been identified in numerous subfractions devoid of CD34 or CD38(Eppert et al., 2011). Observations of differential DR expression innormal and AML human hematopoietic samples strongly suggest the humanAML LSCs are heterogeneous and drug targeting should be based onmolecular pathways instead of surrogate phenotype predications.

Aside from hematopoietic tissue, somatic CSCs have recently beenidentified and validated in human breast tumors and have a CD44+CD24−/lophenotype (Al-Hajj et al., 2003). Using primary human breast tumorswhich test negative for estrogen receptor (ER−), progesterone receptor(PR−), and human epidermal receptor 2 (HER2−) that are associated withthe poorest prognostic outcomes (Dent et al., 2007) we reveal DRcolocalization on the CD44+CD24−/lo breast CSCs (n=3 patients) (FIGS. 14e-f and FIG. 15 h). This finding is consistent with the low levels ofDRs found in normal mammary gland tissue, whereas benign breast tumorsshow intermediate levels and breast cancers display high levels of thesereceptors (Carlo, 1986). Whether the DR expression in AML-blasts wascorrelative to incidence of LSCs in AML patients was investigated. AMLsamples with a large fraction of DRD3+ blasts (FIG. 14 g) and DRD5+blasts (FIG. 14 h) contain LSCs as they are able to initiate leukemia inxenotransplantation recipients, unlike AML patient samples withsignificantly lower levels of DRs that do not contain LSCs. Samples fromAML patients containing LSCs have been correlated to poor prognosticoutcome while non-LSC samples demonstrate a good prognosis (Eppert etal., 2011). High levels of DR expression correlate with poor prognosiswhile low levels demonstrate good prognosis (FIG. 14 g-h) suggestingthat DR assessment has prognostic biomarker applications and is lesscomplex than molecular signatures or LSC readouts for each AML patient.Based on initial identification in neoplastic hPSCs, these collectiveresults suggest a potentially more generalizable role for DR expressionin human somatic CSCs than anticipated, and validate DR as a candidatebiomarker for other CSCs in the human.

Example 12 Thioridazine Antagonism of DR Inhibits Human AML

To better understand the functional role of DR in human AML, two AMLcell lines derived from patients; AML-OCI2 and AML-OCI3, were utilized(Koistinen et al., 2001).

Like primary samples, these two cell lines revealed expression for eachDR1-5 (FIG. 16 a) at markedly higher levels than seen in patientsamples. Due to the bioavailability of dopamine in fetal bovine serum(FBS) (Little et al., 2002), serum-free conditions were employed toassess the role of DRs in AML. Both AML lines were treated withthioridazine and compared to other known DR antagonists clozapine andchlorpromazine (Seeman and Lee, 1975). All three DR antagonists reducedthe number of AML cells upon treatment (FIG. 16 b). To further evaluatethe specificity of DR targeting on human AML cells, patient AML sampleswere divided into DR+ and DR− subfractions using fluorescence activatedcell sorting before being treated with DMSO vehicle or thioridazine for24 h and then assayed for blast-CFU content. A reduction in blast-CFUgeneration was only observed in the DR+ subfraction treated withthioridazine (FIG. 17 a) whereas no reduction was observed in DR−subfraction treated with thioridazine (FIG. 17 b). Conversely, theaddition of a DR D2-family agonist, 70H-DPAT, increased the number ofAML cells (FIG. 16 c). DR D2-family and D1-family exert opposing actionson intracellular signaling leading to differential biological effects(Self et al., 1996). Treatment with a DR D1-family agonist, SKF38393,resulted in a significant reduction in AML cell number confirming thatD2-family signaling is necessary for AML cell survival (FIG. 16 d).These combined results suggest the mechanism of thioridazine's action isthrough antagonism of D2-family DRs and not due to off-target effects,and identifies a novel avenue of CSC targeting via DR signaling.

While the present disclosure has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the disclosure is not limited to the disclosed examples.To the contrary, the disclosure is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

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1. A method of treating acute myeloid leukemia (AML) in a subjectcomprising administering to the subject a dopamine receptor antagonist.2. The method of claim 1, wherein the dopamine receptor antagonist is aD₂ family dopamine receptor antagonist.
 3. The method of claim 2,wherein the dopamine receptor antagonist is a phenothiazine derivativeor a compound listed in Table
 1. 4. The method of claim 3, wherein thephenothiazine derivative is thioridazine.
 5. The method of claim 1,wherein the acute myeloid leukemia comprises cells that express one ormore dopamine receptors (DR) selected from DR1, DR2, DR3, DR4 and DR5.6. The method of claim 5, wherein the cells that express one or moredopamine receptors also express CD14.
 7. The method of claim 1, whereinthe dopamine receptor antagonist preferentially induces thedifferentiation of cancer stem cells relative to hematopoietic or normalstem cells.
 8. The method of claim 1, wherein the subject is inremission.
 9. A method for reducing the proliferation of a cancer cellcomprising contacting the cancer cell with a dopamine receptorantagonist.
 10. The method of claim 9, wherein the cancer cell is acancer stem cell and contacting the cancer stem cell with the dopaminereceptor antagonist induces differentiation of the cancer stem cell. 11.The method of claim 9, wherein the cancer cell is an acute myeloidleukemia (AML) cell.
 12. The method of claim 9, wherein the cell is invivo or in vitro.
 13. The method of claim 9, wherein the dopaminereceptor antagonist is a D₂ family dopamine receptor antagonist.
 14. Themethod of claim 9, wherein the dopamine receptor antagonist is aphenothiazine derivative or a compound listed in Table
 1. 15. The methodof claim 14, wherein the phenothiazine derivative is thioridazine.
 16. Amethod of identifying a subject with cancer suitable for treatment witha dopamine receptor antagonist, the method comprising determining theexpression of one or more dopamine receptors in a sample of cancer cellsfrom the subject, whereby subjects with cancer cells that express one ormore dopamine receptors are identified as suitable for treatment withthe dopamine receptor antagonist.
 17. The method of claim 16, whereinthe step of determining the expression of one or more dopamine receptorsin the sample comprises testing the sample for levels of polynucleotidesor polypeptides that code for one or more dopamine receptors.
 18. Themethod of claim 16, wherein the one or more dopamine receptors areselected from DR1, DR2, DR3, DR4 and DR5.
 19. The method of claim 18,wherein the one or more dopamine receptors include D₂ family dopaminereceptors.
 20. The method of claim 16, wherein the cancer is leukemia orbreast cancer.
 21. The method of claim 20, wherein the cancer is acutemyeloid leukemia or monocytic leukemia.
 22. A method of determining aprognosis for a subject with cancer, the method comprising: determininga level of expression of one or more biomarkers selected from dopaminereceptor (DR) 1, DR2, DR3, DR4 and DR5 in a sample from the subject, andcomparing the level of expression of the one or more biomarkers to acontrol, wherein an increased level of expression of the one or morebiomarkers relative to the control indicates that the subject has a moresevere form of cancer.
 23. The method of claim 22, wherein the step ofdetermining the expression of one or more biomarkers in the samplecomprises testing the sample for levels of polynucleotides orpolypeptides that code for the one or more biomarkers.
 24. The method ofclaim 22, wherein the biomarkers are DR3 and/or DR5.
 25. The method ofclaim 22, wherein the cancer is leukemia and an increased expression ofone or more biomarkers compared to the control indicates a more severeform of leukemia.
 26. The method of claim 25, wherein the leukemia isacute myeloid leukemia or monocytic leukemia.
 27. A method foridentifying a subject with leukemia comprising: determining a level ofexpression of one or more biomarkers selected from dopamine receptor(DR) 1, DR2, DR3, DR4 and DR5 in a sample of white blood cells from thesubject, and comparing the level of expression of the one or morebiomarkers to a control.
 28. The method of claim 27, wherein anincreased level of expression of one or more dopamine receptors comparedto the control is indicative of a subject with leukemia.
 29. The methodof claim 28, wherein the leukemia is acute myeloid leukemia or monocyticleukemia.
 30. A method of screening compounds for anti-cancer activitycomprising: identifying compounds that antagonize one or more dopaminereceptors, wherein compounds that antagonize dopamine receptors areidentified as having anti-cancer activity.
 31. The method of claim 30,wherein the anti-cancer activity comprises reduced proliferation ofbreast cancer cells, AML cells or monocytic cells.
 32. A method foridentifying a cancer stem cell from a population of cells, the methodcomprising: determining whether a cell expresses one or more biomarkersselected from dopamine receptor (DR) 1, DR2, DR3, DR4 and DR5; whereinexpression of the one or more biomarkers is indicative that the cell isa cancer stem cell.
 33. The method of claim 32, wherein the populationof cells comprises cells isolated from a mammal or cells in tissueculture.
 34. The method of claim 32, wherein the population of cellscomprises pluripotent stem cells.
 35. The method of claim 32, whereinthe population of cells comprises cancer cells or pre-cancerous cellssuch as leukemic cells or breast cancer cells.
 36. The method of claim32, wherein the population of cells comprises hematological cancercells.
 37. The method of claim 32, wherein the step of determiningwhether the cell expresses one or biomarkers comprises testing the cellfor the expression of polynucleotides or polypeptides that code for DR1,DR2, DR3, DR4 or DR5.
 38. The method of claim 32, wherein a cell thatexpresses DR1, DR2, DR3, DR4 and DR5 is identified as a cancer stemcell.
 39. The method of claim 32, further comprising isolating thecancer stem cells from the population of cells.
 40. The method of claim39, wherein the step of isolating the cancer stem cells form thepopulation of cells comprises flow cytometry, fluorescence activatedcell sorting, panning, affinity column separation, or magneticselection.